Volume 22, Issue 1, Pages 33-39 (January 2012) Serotonin Signaling Is Required for Wnt-Dependent GRP Specification and Leftward Flow in Xenopus  Tina Beyer, Michael Danilchik, Thomas Thumberger, Philipp Vick, Matthias Tisler, Isabelle Schneider, Susanne Bogusch, Philipp Andre, Bärbel Ulmer, Peter Walentek, Beate Niesler, Martin Blum, Axel Schweickert  Current Biology  Volume 22, Issue 1, Pages 33-39 (January 2012) DOI: 10.1016/j.cub.2011.11.027 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Serotonin Signaling Is Required for the Development of Leftward Flow (A and B) Altered Pitx2c mRNA expression patterns and quantification of results in stage (st) 28–33 xHtr3 morphants and LBD-injected specimens. The following abbreviations are used: wt, left-sided; bi, bilateral. (C) Leftward flow in dorsal explants of stage 17 embryos. Quantification of flow quality (rho) is shown, as calculated from time-lapse movies. (D–J) GRP analysis. (D–G) SEM photographs of GRP areas in wild-type embryo (D), MO1 morphant (E), MO1 + xFL mRNA (F), and xLBD-injected specimen (G). Coloration indicates nonciliated cells (red), cells with normal posteriorly polarized cilia (green), and cells with cilia in other locations (blue). Insets show cilia in higher magnification (arrowheads). Scale bar represents 25 μm. (H–J) Quantification of ciliation rate (H), relative cilia length (I), and cilia polarization (J) of GRP cilia in defined areas, indicated by white squares in (D)–(G). “x” in box plots represents mean values. The following abbreviations are used: a, anterior; l, left; p, posterior; r, right; ∗, significant; ∗∗, highly significant; ∗∗∗, very highly significant; n.s., not significant. Numbers in parentheses indicate number of embryos or dorsal explants analyzed. Statistical significance was calculated using Pearson's chi-square test (B) or Student's t test (C and H–J; [43]). See also Figure S1. Current Biology 2012 22, 33-39DOI: (10.1016/j.cub.2011.11.027) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Serotonin Signaling Is Required for SM Specification Expression of SM marker genes Foxj1 (A and G) and Xnr3 (D and H) was downregulated in MO1 morphants (B, E, G, and H) and xLBD- or hLBD-injected specimens (F–H). Coinjection of xFL rescued Foxj1 mRNA levels (C and G). (A–H) Vegetal view, dorsal side up (A–E); dorsal view, animal side up (F). Quantification and statistical analysis of results (G and H). Numbers in parentheses represent number of assessed embryos. Statistical significance was calculated using Pearson's chi-square test [43]. The following abbreviation is used: ∗∗∗, very highly significant. See also Figure S2. Current Biology 2012 22, 33-39DOI: (10.1016/j.cub.2011.11.027) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Interplay of Serotonin and Canonical Wnt Signaling (A and B) Induction of complete or partial secondary (2°) axes by ventral injection of XWnt8 mRNA at the four-cell stage was inhibited by parallel injection of MO1, MO2, xLBD, or hLBD mRNA. Twinning was rescued upon coinjection of xFL and MO1 or upon coinjection of xLBD or hLBD and serotonin. (C and D) No inhibition of dvl2 (C) or XSia (D) induced 2° axis formation in serotonin signaling-impaired embryos. (E–G) Reduction of Foxj1 expression in morphants was rescued by coinjection of dvl2 (E) or β-cat (F) mRNA. Gastrula embryos at stage 10–11.5 in vegetal view (dorsal side up) are shown. (G) Quantification of results. Numbers in parentheses represent number of embryos analyzed. Statistical significance was calculated using Pearson's chi-square test [43]. The following abbreviations are used: ∗∗, highly significant; ∗∗∗, very highly significant; n.s., not significant. See also Figure S3. Current Biology 2012 22, 33-39DOI: (10.1016/j.cub.2011.11.027) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Symmetric Serotonin Localization from Early Cleavage Stages through Gastrulation Suggests a Role as Competence Factor for Canonical Wnt Signaling (A) Experimental setup. Embryos injected at the four-cell stage with 20 ng serotonin into single ventral or dorsal blastomeres (B–E) or uninjected embryos (F–K) were fixed at the stages indicated, bisected equatorially into animal and vegetal halves (B–H) or sagittally (I–K), immunostained for serotonin, and mounted and viewed under a confocal laser-scanning microscope as shown. Z stacks of optical sections were sampled at intervals as described in the Supplemental Experimental Procedures, beginning at the first section beyond the uneven surface of the cut. Images in (B)–(F) and (I) are maximum projections of z stacks. (B–E) Serotonin-injected embryos at the 32-cell stage (B and D) and 256-cell stage (C and E). Note that serotonin localization was confined to the injected cell lineage throughout the culture period, i.e., did not spread or accumulate elsewhere (inset in D). (F–H) Endogenous distribution of serotonin at the 32-cell stage. In the equatorial section cut just below the blastocoel floor (F), two general localizations of serotonin are seen in each blastomere: a thick cortical layer (G) and a perinuclear cloud (H). The inset in (G) shows a higher magnification to demonstrate the punctate nature of cytoplasmic serotonin staining. (I–K) Endogenous distribution of serotonin at the onset of gastrulation (stage 10). Although serotonin is found in every cell as at earlier stages, it becomes enriched in the superficial layer of the animal cap (I and J). The contrast in serotonin content between the future floor and roof of the archenteron is evident by the abrupt transition seen at the dorsal lip (K). Serotonin staining in red; green color represents autofluorescence (yolk). The following abbreviations are used: an, animal; bc, blastocoel; d, dorsal; dc, deep cells; v, ventral; veg, vegetal. Asterisk highlights nucleus. (L) Role of serotonin signaling in LR axis specification: a model. Schematic representation of serotonin localization at blastula stage. Serotonin has accumulated in the superficial animal epithelium of the blastula embryo and mediates competence for canonical Wnt signaling to specify the precursor of the GRP in the dorsal superficial layer, i.e., the SM. Differences in serotonin concentrations indicated by color code. Wnt signaling is depicted by the nuclear β-cat gradient as black dots of differing intensity [42]. Lack of Wnt signaling on the ventral side precludes SM specification. The following abbreviations are used: an, animal; d, dorsal; st. 9, stage 9; v, ventral; veg, vegetal. For details see text. See also Figure S4. Current Biology 2012 22, 33-39DOI: (10.1016/j.cub.2011.11.027) Copyright © 2012 Elsevier Ltd Terms and Conditions